Structure-activity relationships in platelet-activating factor. 12. Synthesis and biological evaluation of platelet-activating factor antagonists with anti-HIV-1 activity

Article abstract of DOI:10.1021/jm040860g

The HIV-1 central nervous system infection leads to the onset of neurological impairments called AIDS dementia complex (ADC). PAF plays an important role in this pathology, as it is an HIV-1-induced neurotoxin produced by infected or activated macrophages

Full text of DOI:10.1021/jm040860g

6410
J. Med. Chem. 2004, 47, 6410-6419
Structure-Activity Relationships in Platelet-Activating Factor. 12. Synthesis
and Biological Evaluation of Platelet-Activating Factor Antagonists with
Anti-HIV-1 Activity
Nawal Serradji,† Marc Martin,‡ Okkacha Bensaid,†,§ Salvatore Cisternino,| Christophe Rousselle,|
Nathalie Dereuddre-Bosquet,‡, Jack Huet,† Catherine Redeuilh,† Aazdine Lamouri,† Chang-Zhi Dong,†
Pascal Clayette,‡, Jean-Michel Scherrmann,| Dominique Dormont,‡ and Franc¸oise Heymans*,†
Unite´ de Recherche Pharmacochimie Mole´culaire et Syste`mes Membranaires, Laboratoire de Pharmacochimie Mole´culaire,
Universite´ Paris 7sDenis Diderot, case 7066, 2 Place Jussieu, 75251 Paris Cedex 05, France; CEA, Service de Neurovirologie,
DSV/DRM, CRSSA, EPHE, Universite´ Paris-Sud Orsay, 18 route du Panorama, BP 6, 92265 Fontenay aux Roses, France;
SPI-BIO c/o Service de Neurovirologie, CEA, Fontenay aux Roses Cedex, France; INSERM U26, Hoˆpital Fernand Widal,
200 rue du Faubourg Saint-Denis, 75475 Paris Cedex 10, France; and COSNA, Laboratoire de Chimie Organique,
Substances Naturelles et Analyses, Faculte´ des Sciences, Universite´ Aboubakr Belkaid, B.P 119, Tlemcen, Alge´rie
Received July 8, 2004
The HIV-1 central nervous system infection leads to the onset of neurological impairments
called AIDS dementia complex (ADC). PAF plays an important role in this pathology, as it is
an HIV-1-induced neurotoxin produced by infected or activated macrophages and microglia,
in the brain. We previously reported that PAF-antagonists bearing a trisubstituted piperazine
presented in vitro anti-HIV-1 activity in human macrophages. To improve the pharmacological
activities of our lead compound, 1a, we modified its carbamate function and evaluated both its
antiretroviral and anti-PAF activities. One carbamate derivative (10c) demonstrated a similar
antiviral activity but a higher anti-PAF potency, whereas 4a, with an ureide function, presents
an increased antiviral activity and can be considered as a pure antiretroviral drug, as it does
not present PAF-antagonism. Moreover, we measured the ability of 1a to cross the blood-
brain barrier, using the in situ mouse brain perfusion method and its plasmatic concentrations
after iv and po administration. The transport parameter measured (Kin) proves that 1a is able
to cross this biological barrier, but a pharmacokinetic study reveals its weak bioavailability in
rats.
Introduction
synthesis in HIV-infected cells of monocytic lineage.8,9
High levels of PAF are detected in cerebrospinal fluids
(CSF) of patients with neurologic dysfunctions and
correlated with the degree of neurological impairments.
Moreover, when added at concentrations close to those
found in CSF of HIV-infected patients, PAF produces a
dose-dependent neurotoxicity, inhibited by NMDA un-
competitive antagonists MK-801 and memantine.7 The
neurotoxicity induced by HIV-1-infected macrophages
can be prevented by PAF-acetylhydrolase (PAF-AH), the
main enzyme responsible of PAF catabolism.10
Even if the virus plasma level falls below the limit of
detection (20-500 HIV RNA copies/mL, depending on
the assay used) and neurological disorders are markedly
improved in 50-60% of the patients treated by highly
active antiretroviral therapy (HAART), the reappear-
ance of HIV in the blood of these patients suggests that
this treatment may not eradicate the virus in certain
reservoirs, such as the CNS, where it is rapidly repli-
cated and delivered to the periphery. This hypothesis
is supported by the weak penetration or the efflux of
actual antiretroviral drugs into the brain.11
The infection of central nervous system (CNS) by the
human immunodeficiency virus (HIV) occurs early after
the systemic infection1 and leads to the onset of neuro-
logical dysfunctions named acquired immune deficiency
syndrome (AIDS) dementia complex (ADC), observed in
15-20% of patients.2,3 Productive replication of HIV-1
in brain macrophages and microglia is a critical com-
ponent of viral pathogenesis. However, how virus-
macrophage interactions lead to neurological disease
remains incompletely understood.4 Neither, it is clear
whether ADC results from the trafficking into the brain
of infected or activated monocytes or direct infection of
the brain by the virus. Nevertheless, there is a consen-
sus to say that secretory products from HIV-1-infected
macrophages are the likely source of neurotoxic activi-
ties. Indeed, after an antigenic stimulation in vitro or
contact with neural cells in vivo, infected monocytes
release high levels of the proinflammatory cytokine
tumor necrosis factor-R (TNF-R) and the phospholipidic
mediator platelet-activating factor (PAF).5-7 HIV-1
production is up-regulated by TNF-R in infected mac-
rophages, and PAF, in turn, appears to increase TNF-R
Taking together, these results suggest that PAF plays
an important role in the HIV-1-induced neurologic
dysfunctions and prompted us to evaluate, in our
previous publication, the antiviral activity of PAF
antagonists. This led us to the discovery of our lead
compound, PMS 601 (compound 1a previously de-
scribed,12 Figure 1), a trisubstituted piperazine that is
* To whom correspondence should be addressed. Tel: 33 1 4427
6050. Fax: 33 1 4427 5641. E-mail: heymans@ccr.jussieu.fr.
† Universite´ Paris 7sDenis Diderot.
‡ CEA.
§ Universite´ Aboubakr Belkaid.
| INSERM U26.
SPI-BIO.
10.1021/jm040860g CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/10/2004

PAF Antagonists with Anti-HIV-1 Activity
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6411
Results and Discussion
Evaluation of New 1a Derivatives. All the com-
pounds were tested for their ability to inhibit PAF-
induced platelet aggregation on one hand and for their
activity in MDM infected with the reference macro-
phage-tropic HIV-1/Ba-L strain on the other hand,12,13
and the activities are reported in Table 1.
Figure 1. Compound 1a (PMS 601).
In these experiments, 1a demonstrated identical
effects as previously described with an anti-HIV IC50
and an anti-PAF IC50 equal to 11 and 8 µM, respec-
tively.12
a PAF antagonist (8 µM IC50) and possesses anti-HIV-1
activity (11 µM IC50). It has been shown that it is able
to diminish both HIV-1 replication and the neurotoxic
effects induced by the important production of PAF by
the infected cells in brain (data not shown).12,13 To
improve the biological activities of 1a, we modified the
carbamate linkage to ureate and reversed carbamate
and thionocarbamate. Different alkyl substituents of the
above functions were used to maintain either the
structure skeleton or lipophilicity. Some less lipophilic
ester or monosubstituted carbamate derivatives than
what have been published12 are also described in this
work to complete this structure-activity relationship
study.
We verified whether 1a, the most studied compound
of the series, is able to cross the blood-brain barrier
(BBB), using the in situ brain perfusion technique, and
reach the virus in one of its main reservoirs and
measured its pharmacokinetic parameters in male
Sprague-Dawley rats.
The introduction of an ureide function as in 10a, a
direct analogue of 1a, leads to the loss of both anti-PAF
and anti-HIV activities, apparently due to the log P
decrease (fW ) -0.564) or the presence of a new NH
moiety. To distinguish which is the main factor respon-
sible of this diminution, 10b, an isolipophilic derivative
of 1a, was synthesized and tested. The result shows that
this compound remains less active than our lead in both
biological assays. Taken together with the result of 1d,
the carbamate analogue of 10b previously described,12
it seems clear that the new NH moiety induces a drastic
influence on the anti-PAF activity, as already proposed
in our previous publication for an amide analog,12 and
suggests that other factors than lipophilicity are in-
volved. Indeed, we cannot exclude an intramolecular
H-bond between the ureide linkage and the amide group
or between the compound and its biological target. This
could modify, especially in the first case, the “cache-
oreilles” effect and prevent the molecule from interact-
ing efficiently with the PAF-R.
Chemistry
By action of commercially available isocyanates on the
amine 1, reported by Serradji et al.,12 urea derivatives
2a,b were synthesized following method A in Scheme
1, while the carbamate 2c was prepared by condensing
isopropyl chloroformate with the amine 1. The phenyl
carbamate 7 and the ureide 6 were obtained from the
condensation of phenyl chloroformate on the alcohol 5,
as previously described,14 and the amine 1, respectively.
N,N-Disubstituted ureides 8a,b were obtained by treat-
ment of phenyl carbamate 6 (method B, Scheme 1) with
the corresponding amines and 8c by action of the
appropriate amine on the phenyl carbonate 7. The
benzyl groups of 2a-c and 8a-c were removed by
catalytic hydrogenolysis to afford the corresponding
2-substitued piperazines 3a-c and 9a-c, which, when
treated with 3,4,5-trimethoxybenzoyl chloride, led to
4a-c and 10a-c.
As outlined in Scheme 2, the ester 12 was derived
from the previously described compound 11 by deben-
zylation.14 The piperazinic nitrogens were then substi-
tuted using 3,4,5-trimethoxybenzoyl chloride, and the
ester 13 thus obtained was reduced into the correspond-
ing alcohol 14 by sodium borohydride in MeOH. The
thionocarbamoyl functions (15a,b) were introduced by
condensation of the alcoholate of 14 with the commercial
thiocarbamoyl chloride, while ester 15c was synthesized
by reacting 14 with propanoyl chloride.
We have shown in our previous publication12 that the
replacement of the N,N-diethylamino moiety by a pyr-
rolidino or a piperidino group in the C-substituent of
1a had no influence or increased its potency on the
PAF-R, while its anti-HIV capacity was reduced. How-
ever, using the N-monoalkylamino group with a weak
steric hindrance of the alkyl chain close to the carbonyl
function decreased less the antiviral activity. This
prompted us to synthesize compound 4a, in which a
linear hexyl was used and whose lipophilicity is similar
to that of 10b. It is worthwhile to note that this
compound is not only much more active than 10b but
also 10-fold more potent than 1a against HIV, although
its anti-PAF property is not improved. This underlines
the importance of the second NH for the antiviral
activity in these isolipophilic compounds. The decrease
of this activity for 4b (IC50 ) 32 µM) could be explained
by the steric effect generated by the o-methyl substit-
uents on the phenyl group.
However, compared to 1a, only the anti-PAF activity,
but not at all the antiviral potency, was improved
when an n-butyl group was used, instead of two ethyls,
in the carbamate moiety to preserve a similar global
lipophilicity, as demonstrated by the biological data of
10c.
Moreover, when the nitrogen and the oxygen atoms
are inverted (compound 4c), the anti-PAF activity is
unchanged and the anti-HIV activity is weakly dimin-
ished. This suggests that the order of the atoms in the
carbamate function would not be important to keep both
activities.
Tritylation followed by acylation with 3,4,5-trimethoxy-
benzoyl chloride of 1614 afforded 18 through the inter-
mediate 17. An acid deprotection of 18 into 19 and
condensation of the latter with syringic acid using DCC
led to 20, as described in Scheme 3. [3H]1a (compound
21, Scheme 3) was prepared by alkylation of compound
20 with commercial [3H]methyl iodide (Amersham,
France).
The ester 15c (fW ) 0.488) was synthesized to
complete our preliminary results, since the ester ana-

6412 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Serradji et al.
Scheme 1a
a Reagents: (a) OdCdN-R, Et2O or RCOCl, Et3N, toluene: (b) H2, Pd/C (10%), EtOH/HCl or CH3COOH, 40 °C; (c) 3,4,5-(MeO)3PhCOCl,
Et3N, CH2Cl2. b Reagents: (a) PhOCOCl, Pyr, CH2Cl2, 0 °C; (b) HN(R1R2), reflux; (c) H2, Pd/C (10%), EtOH/HCl or CH3COOH, heat; (d)
3,4,5-(MeO)3PhCOCl, Et3N, CH2Cl2.
Scheme 2a
a Reagents: (a) H2, Pd/C, EtOH, heat; (b) 3,4,5-(MeO)3PhCOCl, Et3N, CH2Cl2; (c) NaBH4, MeOH, 0 °C; (d) YCl, NaH, DMF, 70-80 °C
or Et3N, CH2Cl2.
logue (W ) CH2OCOCMe3) previously described12 pos-
sesses a potent anti-PAF activity but did not present a
good antiviral one. The result obtained with 15c shows
clearly that this lack should not be due to the high
lipophilicity of pivalate (fW ) 1.526), as 15c is isolipo-
philic to 1a (fW ) 0.488), and that the ester bond is not
favorable for the antiviral activity.
When the carbonyl function in the C-substituent of
1a was changed to a thiocarbonyl as in 15a, an increase
of the lipophilicity was not associated with an augmen-
tation of the anti-PAF activity as expected. In addition,
with this modification, the molecule becomes highly
toxic on MDM at 10 µM and is inactive at 1 µM. Thus,
its antiviral activity could not be evaluated. The 1a
isolipophilic compound 15b (×c4W ) 0.611) presents a
higher anti-PAF potency but a moderate antiviral
activity, showing that the introduction of a sulfur atom
in this substituent does not improve both of them.
Blood-Brain Barrier Permeability of 1a. One of
the main defects of HAART to eradicate the virus seems
to be related to the weak penetration of these drugs into
certain tissues, such as the CNS, which, in consequence,
could be considered as a virus reservoir. New antiviral
agents capable to cross the BBB efficiently represent
potential candidates to struggle against HIV toward a
complete eradication of the virus. This promped us to
evaluate the intrinsic permeability of 1a into the brain,
the most studied compound of the series.12 This evalu-
ation was performed in mice using [3H]1a and the in
situ brain perfusion method.15
Cerebral vascular volume was preserved, and thus,
the BBB integrity was maintained during the perfusion,
as shown by the fact that no significant penetration was
observed when radioactive [14C]sucrose, a marker of
brain vascular volume, was coperfused during brief
periods.

PAF Antagonists with Anti-HIV-1 Activity
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6413
Scheme 3a
a Reagents: (a) Ph3CCl, Et3N, CH2Cl2; (b) 3,4,5-(MeO)3C6H2COCl, Et3N, CH2Cl2; (c) HCl, MeOH; (d) 4-OH-3,5-(MeO)2C6H2COOH, DCC,
HOBT, CH2Cl2, reflux; (e) [3H]CH2Cl, K2CO3, CH3CN/toluene.
The blood transport coefficient of [3H]1a (Kin ) 1.02
( 0.05 µL/s/g), i.e., its brain uptake, obtained is not only
higher than that of an hydrophilic compound such as
urea, one of the least permeable compounds of the BBB
(Kin ≈ 0.1 µL/s/g), but also than that of morphine (Kin
) 0.26 ( 0.04 µL/s/g), which is a well-known neuroactive
drug.16 This experiment enables us to compare the
capacities of 1a vs morphine to cross the BBB, but it
does not establish its antiviral activity within the brain.
Indeed, morphine acts through its receptor and 1a’s
mode of action is still unknown. This means that 1a has
an intrinsic capacity to cross the biological barrier and,
consequently, is susceptible to inhibit HIV-1 replication
and antagonize PAF activity in the brain, without
changing BBB integrity. However, the brain extraction
using the molecular weight of 1a (MW ) 603 g/mol).
Using this calculation, we showed that the plasma
concentrations of 1a are equal to 141 ( 46 and 26 ( 6
µM after the iv and per os administrations, respectively.
These calculations are between IC50 (20 ( 15 µM) and
IC70 (43 ( 23 µM), and IC70 and IC90 (189 ( 109 µM)
obtained in experiments performed with MDM infected
with different macrophage-tropic strains (data not
shown). It is probable that these concentrations are
insufficient to enable a significant in vivo anti-HIV
activity.
It has been reported very recently that BMS-378806,
a trisubstituted piperazine derivative, is able to inter-
fere with CD4-gp120 interactions.19 It exhibits not only
a good HIV-1 inhibitory profile with high specificity in
a great number of different virus strains but also
interesting pharmaceutical and pharmacokinetic prop-
erties, without crossing the BBB to any appreciable
extent,20 in three animal models. It is evident that this
compound could be considered as a new HIV-1 attach-
ment inhibitor. The important structural similarities of
this series with 1a do not lead to a similar mode of
action, as we know that this compound is not an HIV-1
entry inhibitor in the cells (data not shown).
coefficient of 1a (2.5%), obtained from the ratio (Kin 1a
/
Kin diazepam) × 100, shows clearly that 1a uptake from
the blood to the CNS is not complete, since diazepam,
a lipophilic compound which freely crosses the BBB and
thus was used to estimate the flow rate, has a blood
transport coefficient of 42.5 µL/s/g.16 However, this
evaluation was performed on an intact biological mem-
brane, and the BBB changes during invasion of the CNS
by HIV-1 were not taken into account. Indeed, inflam-
matory and toxic molecules secreted by monocytes and
microglia as well as viral proteins such as gp120, Tat,
and Nef seem to be involved in the modifications of the
functional integrity of tight junctions of brain endothe-
lium.17,18 On the other hand, the multidrug resistance
pump P-glycoprotein (P-gp) has been identified as an
important determinant of drug permeability across the
BBB.16 Thus, the potential influence of this protein on
the penetration of 1a through the BBB is also to be
evaluated.
Conclusion
On the basis of our previous work, we have demon-
strated in this study that using a n-butyl instead of
diethyl moiety on the carbamate function only increases
the anti-PAF activity. However, when the carbamate
function is replaced by an ureide one and a linear chain
is incorporated to maintain the lipophilicity, the anti-
viral activity is highly enhanced. This opens new
perspectives in the development of original antiviral
agents. Moreover, 1a’s Kin and log Poctanol/water (log
Poctanol/water ) 0.8 vs log Poctanol/saline ) 1.02 ( 0.02 for 1a
and [3H]AZT,21 respectively), its ability to increase AZT
activity in infected MDM, and its lack of toxicity in rats
after oral administration (LD50 > 2000 mg/kg without
mortality at this dose level, unpublished data) justify
its use in the search for new derivatives with increased
antiviral activity and bioavailability.
Pharmacokinetic Study of 1a. The objective of this
investigation was to determine the pharmacokinetic
profiles of 1a after its single administration by intra-
venous and oral routes to male Sprague-Dawley rats.
The bioavailability was also calculated. The main phar-
macokinetic parameters (calculated for a dose of 1
mg/kg) are summarized in Table 2. To appreciate the
anti-HIV efficacy of maximal 1a concentrations found
in plasma of rats, the micromolar values were calculated

6414 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Serradji et al.
Table 1. Influence of Substituent W on in Vitro Anti-HIV and Anti-PAF Activities
a Lipophilic contribution of W calculated from Rekker et al.23 b Antiviral activity determined with HIV-1/Ba-L-infected monocyte-
derived macrophages, from a single measurement (see the Experimental Section). c Inhibition of PAF-induced platelet aggregation using
platelet-rich plasma (PRP) of New Zealand rabbits calculated from a dose-response curve as described in the Experimental Section (n )
5, mean ( 10%). d Fifty percent cytotoxicity concentration evaluated by neutral red staining. e See Serradji et al.12 f Percent inhibition
of HIV-1 replication at 100 µM. g Not determined because highly toxic at 10 µM.
Table 2. Pharmacokinetic Parameters for 1a in Ratsa,b
TLC plastic sheets of silica gel 60F254 (layer thickness 0.2
mm) from Merck. Column chromatography purification was
carried out with silica gel 60 (70-230 mesh ASTM, Merck).
All melting points were determined on a digital melting point
apparatus (Electrothermal) and are uncorrected. The struc-
tures of all compounds were confirmed by IR and 1H NMR
spectra. IR spectra were obtained in paraffin oil with a ATI
Mattson Genesis Series FTIR spectrometer, and 1H NMR and
13C NMR spectra were recorded in CDCl3 or in DMSO-d6 on
a BRUCKER AC 200 spectrometer using hexamethyldisi-
loxane (HMDS) as an internal standard. Chemical shifts are
given in ppm and peak multiplicities are designated as fol-
lows: s, singlet, d, doublet, dd, doublet doublet, t, triplet, br
s, broad singlet, m, multiplet, q, quadruplet, sex, sextuplet,
sep, septuplet. Elemental analyses were obtained from the
“Service Re´gional de Microanalyse” (Universite´ Pierre et Marie
Curie, Paris, France) and were within (0.4% of theoretical
values.
after iv
administration
after po
administration
Cmax (ng/mL)
t1/2 (h)
861 ( 338
2.14 ( 0.17
1828 ( 716
-
33 ( 12.1
2.29 ( 0.40
177 ( 106
10.9 ( 1.1
AUC (ng‚h/mL)c
bioavailability (%)
a Three animals were included in this study and received each
100 mg/kg by iv route and 500 mg/kg by oral route. b All the results
are normalized for an administration dose of 1 mg/kg of body
weight in order to compare both routes of administration. c Area
under curve.
Experimental Section
Chemistry. General Methods. All materials were ob-
tained from commercial suppliers and used without further
purification. Thin-layer chromatography was performed on

PAF Antagonists with Anti-HIV-1 Activity
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6415
1,4-Dibenzyl-2-(hexylaminocarbonylaminomethyl)-
piperazine (2a). A solution of hexyl isocyanate (1.73 g, 13.56
mmol) in ether (50 mL) was added dropwise to a solution of
the amine 1 (4 g, 13.56 mmol) in ether (50 mL). The solution
was stirred for 2 h at room temperature, the solvent was
eliminated in a vacuum, and the residue was chromatographed
on silica gel column using MeOH/CH2Cl2 (3:97, v/v) as eluent
to yield 2a (5.26 g, 92%) as a wax: Rf 0.15 (MeOH/CH2Cl2, 5:95,
v/v); IR (ν cm-1) 3356 (NH), 3061 (ArH), 1629 (CdO amide),
6H, CH3 acetate); 13C NMR (DMSO-d6) δ 173.64 (CdO acetic
acid), 156.33 (CdO ureate), 136.15, 135.59, 127.61, 125.63
(ArCdC), 53.68 (CH piperazine), 46.42, 43.16, 42.94 (CH2
piperaine), 41.91 (CH2 ureate), 22.69 (CH3 acetic acid), 18.29
(CH3).
2-(Isopropyloxycarbonylaminomethyl)piperazine, Di-
hydrochloride (3c). This compound was prepared from 2c
using the same process as for 3a but with 12 N HCl instead
of CH3COOH, as a solid (4.2 g, 87%): Rf 0.60 (NH4OH/MeOH/
CH2Cl2, 2:20:80, v/v/v); mp 237 °C; IR (ν cm-1) 1712 (CdO);
1H NMR (DMSO-d6) δ 10.01 (br s, 4H, NH2+), 4.72 (t, 1H, J )
6 Hz, NHCdO), 4.00-2.60 (m, 9H, CH2N, CH2 piperazine),
1.14 (d, 6H, J ) 5.6 Hz, CH3); 13C NMR (DMSO-d6) δ 156.28
(CdO), 67.54 (OCH), 51.67 (CH piperazine), 42.08 (CH2NH),
22.03 (CH3).
1
1573 (ArCdC); H NMR δ 7.19-7.06 (m, 10H, ArH), 5.31 (br
s, 1H, NH), 4.99 (t, 1H, J ) 5.5 Hz, NH), 3.92 (d, 1H, J ) 13
Hz, CH-Ph), 3.50-2.81 (m, 4H, CH2NCdONCH2), 3.38 (d, 1H,
J ) 13 Hz, CH-Ph), 3.32 (d, 1H, J ) 13 Hz, CH-Ph), 3.14 (d,
1H, J ) 13 Hz, CH-Ph), 2.55 (m, 4H, piperazine), 2.13 (m, 3H,
piperazine), 1.37 and 1.19 (m, 8H, (CH2)4), 0.77 (t, 3H, J ) 6.4
Hz, CH3); 13C NMR δ 158.68 (CdO), 138.13, 137.59, 128.91,
128.83, 128.10, 126.95, 126.89 (ArCdC), 62.81, 57.54, 55.78,
52.12, 50.03 (CH2 benzyl and piperazine), 58.42 (CH pipera-
zine), 40.45, 39.67, 31.41, 30.11, 26.46, 22.43 (CH2), 13.89
(CH3).
1,4-Dibenzyl-2-((2,6-dimethylphenyl)aminocarbonyl-
aminomethyl)piperazine (2b). The process was the same
as described for 2a but using 2,6-dimethylphenyl isocyanate
to yield 2b (4.25 g, 71%) as a wax: Rf 0.65 (MeOH/CH2Cl2, 5:95,
v/v); IR (ν cm-1) 3337 (NH), 3023 (ArH), 1639 (CdO amide),
1563 (ArCdC); 1H NMR δ 7.24-7.08 (m, 10H, ArH), 7.07-
7.02 (m, 1H, NH-C6H3), 7.03-6.63 (m, 2H, NH-C6H3), 6.62
(br s, 1H, NH), 5.80 (d, 1H, J ) 13 Hz, CH-Ph), 5.03 (br s,
1H, NH), 3.44-3.25 (m, 4H, CH2-Ph, CH2NHCdO), 2.96 (d,
1H, J ) 13 Hz, CH-Ph), 2.90-2.30 (m, 4H, piperazine), 2.20
(s, 6H, CH3), 2.08-1.90 (m, 2H, piperazine), 1.90-1.70 (m, 1H,
piperazine); 13C NMR δ 157.89 (CdO), 138.18, 137.49, 137.18,
134.17, 128.53, 128.32, 128.08, 127.49, 126.93, 126.69
(ArCdC), 62.88, 57.32, 55.43, 52.08, 50.19 (CH2 benzyl and
piperazine), 58.34 (CH piperazine), 39.49 (CH2), 18.34 (CH3).
1,4-Dibenzyl-2-(isopropyloxycarbonylaminomethyl)-
piperazine (2c). A solution of isopropyl chloroformate (1 M
in toluene, 44 mL) was added dropwise to a solution of 1 (10
g, 33.90 mmol) and Et3N (25 mL) in toluene (100 mL). The
solution was stirred at room temperature for 2 h, diluted with
Et2O (100 mL), washed with a saturated NaHCO3 solution and
water, dried over MgSO4, and filtered, and the solvent was
eliminated under reduced pressure to yield 2c (8.65 g, 67%)
as a wax: Rf 0.4 (MeOH/CH2Cl2, 5:95, v/v); IR (ν cm-1) 1712
(CdO amide), 1602 (ArCdC); 1H NMR δ 7.61 (br s, 4H, ArH),
7.34 (br s, 6H, ArH), 6.50 (br s, 1H, NH), 4.70-2.60 (m, 13H,
CH2-Ph, CH2NCdO, piperazine), 3.41 (m, 1H, J ) 7 Hz,
OCH), 1.14 (br s, 6H, CH3).
2-(Hexylaminocarbonylaminomethyl)piperazine, Di-
acetate (3a). To a solution of the ureate 2a (6 g, 14.21 mmol)
in glacial acetic acid (70 mL) was added 100 mg of Pd/C (10%),
and this mixture was warmed at 40 °C and stirred under
hydrogen atmosphere. After the disappearance of the starting
material (shown by TLC), the suspension was filtered, the
solvents were evaporated, and the residue was crystallized in
acetic acid/ether to give 3a (4.3 g, 83.5%) of as crystals: Rf 0.08
(NH4OH/MeOH/CH2Cl2, 2:20:80, v/v/v); mp 119.8 °C; IR (ν
cm-1) 1732 (CdO acetic acid), 1646 (CdO ureate); 1H NMR
(DMSO-d6) δ 7.48 (br s, 4H, NH2+), 6.38 (br s, 1H, NHCdO),
6.23 (br s, 1H, NHCdO), 3.40-2.30 (m, 11H, piperazine,
CH2NCdO), 1.89 (s, 6H, CH3COO-), 1.70-1.10 (m, 8H, (CH2)4),
0.94 (br s, 3H, CH3); 13C NMR (DMSO-d6) δ 173.81 (CdO acetic
acid), 158.38 (CdO ureate), 53.72 (CH piperazine), 46.36, 43.04
(CH2 piperazine), 41.79, 39.38, 31.13, 30.01, 26.15, 22.16 (CH2
ureate), 22.92 (CH3 acetic acid), 13.95 (CH3).
2-(Hexylaminocarbonylaminomethyl)-1,4-bis(3,4,5-tri-
methoxybenzoyl)piperazine (4a). To a solution of 3a (1.76
g, 4.86 mmol) and Et3N (4.05 mL, 29.16 mmol) in CH2Cl2 (50
mL) was added dropwise a solution of 3,4,5-trimethoxybenzoyl
chloride (2.8 g, 12.15 mmol) in CH2Cl2. The solution was stirred
at room temperature overnight and EtOH (5 mL) was added.
The organic layer was washed with saturated NaHCO3 solu-
tion and water, dried over MgSO4, and filtered, and the solvent
was evaporated. The residue was chromatographed using a
silica gel column with MeOH/CH2Cl2 (1:99, v/v) as eluent to
give, after a crystallization in Et2O/MeOH, 4a (1.04 g, 46%)
as white crystals: Rf 0.3 (MeOH/CH2Cl2, 5:95, v/v); mp 94.8
°C; IR (ν cm-1) 3374 (NH), 1631 (CdO ureate), 1621 (CdO
1
amide), 1584 (ArCdC); H RMN δ 6.61 (s, 2H, ArH), 6.59 (s,
2H, ArH), 5.31 (br s, 1H, NH), 5.10-3.90 (m, 5H, NH, CH2
and CH piperazine), 3.90-3.70 (m, 18H, CH3O), 3.70-2.60 (m,
7H, CH2NCONCH2, CH2 piperazine), 1.50-0.95 (m, 8H,
(CH2)4), 0.77 (t, 3H, J ) 6.4 Hz, CH3); 13C NMR δ 171.43,
170.84 (CdO amide), 158.43, 153.38, 153.28, 138.48, 130.09,
104.49 (ArCdC), 60.86, 60.79, 56.29, 56.21 (CH3O), 40.50,
39.05, 31.40, 30.15, 26.45, 22.46 (CH2 ureate), 13.91 (CH3).
Anal. (C32H46N4O9‚0.75H2O) C, H, N.
2-((2,6-Dimethylphenyl)aminocarbonylaminomethyl)-
1,4-bis(3,4,5-trimethoxybenzoyl)piperazine (4b). This com-
pound was prepared from 3b, using the same process as for
4a, as a solid (890 mg, 17%): Rf 0.3 (MeOH/CH2Cl2, 5:95, v/v);
mp 153.3 °C; IR (ν cm-1) 3374 and 3235 (NH), 1651 (CdO
1
ureate), 1624 (CdO amide), 1586 (ArCdC); H RMN δ 7.10-
6.80 (m, 3H, m- and p-ArH), 6.58 and 6.56 (s, 4H, o-ArH),
5.50-4.90 (m, 6H, NH, CH2 piperazine), 4.85-3.75 (m, 12H,
CH3O), 3.75-2.60 (m, 10H, CH2O, CH2NHCdO, CH2 pipera-
zine), 2.35-1.60 (m, 7H, CH3, NH); 13C NMR δ 171.24, 170.79
(CdO amide), 156.84 (CdO ureate), 153.37, 153.15, 139.49,
139.40, 136.47, 134.03, 130.00, 128.36, 104.67, 104.43
(ArCdC), 60.85, 60.77, 56.29, 55.95 (CH3O), 39.11 (CH2
ureate), 18.17 (CH3). Anal. (C34H42N4O9‚0.75H2O) C, H, N.
2-(Isopropyloxycarbonylaminomethyl)-1,4-bis(3,4,5-
trimethoxybenzoyl)piperazine (4c). This compound was
prepared from 3c, using the same process as for 4a, as a solid
(3 g, 70%): Rf 0.3 (MeOH/CH2Cl2, 5:95, v/v); mp 138 °C; IR (ν
cm-1) 3368 (NH), 2976 (ArCH), 1717 (CdO carbamate), 1622
(CdO amide), 1585 (ArCdC); 1H NMR δ 6.60 (s, 2H, ArH),
6.58 (s, 2H, ArH), 5.00 (br s, 1H, NH), 4.80 (sep, 1H, J ) 6.2
Hz, OCH), 4.60-3.90 (m, 3H, CH2NHCdO, piperazine), 3.81,
3.79 and 3.70 (s, 18H, OCH3), 3.70-2.70 (m, 6H, piperazine),
1.15 (d, 6H, J ) 6.2 Hz, CH3); 13C NMR δ 170.97, 170.67
(CdO amide), 156.22 (CdO carbamate), 153.25, 139.30,
139.18, 130.25, 129.81, 104.21 (ArCdC), 67.94 (OCH), 60.56,
56.03, 55.96 (CH3O), 39.31 (CH2), 21.94, 21.84 (CH3). Anal.
(C29H39N3O10‚0.5H2O) C, H, N.
2-((2,6-Dimethylphenyl)aminocarbonylaminomethyl)-
piperazine, Diacetate (3b). This compound was obtained
from 2b (4.24 g, 19.61 mmol) and glacial acetic acid (20 mL),
following the process used for compound 3a, as white crystals
(3 g, 81%): Rf 0.14 (NH4OH/MeOH/CH2Cl2, 2:20:80, v/v/v); mp
149 °C; IR (ν cm-1) 1733 (CdO acetate), 1644 (CdO ureate),
1,4-Dibenzyl-2-(phenoxycarbonylaminomethyl)pip-
erazine (6). To a cooled solution, in an ice bath, of 1 (1.85 g,
6.27 mmol) and pyridine (1 mL, 12.54 mmol) in CH2Cl2 (20
mL) was added dropwise a solution of phenylchloroformate (1.2
mL, 9.40 mmol) in CH2Cl2 (5 mL). The solution was stirred at
0 °C for 1 h and for 3 h at room temperature. It was then
washed with a saturated NaHCO3 solution and water, dried
(MgSO4), and filtered, and the solvent was evaporated in a
vacuum. The residue was chromatographed using a silica gel
1
1569 (ArCdC); H NMR (DMSO-d6) δ 7.91 (br s, 4H, NH2+),
7.00 (br s, 3H, ArH), 6.70 (br s, 1H, NHCdO), 3.40-2.30 (m,
9H, CH2NHCdO, CH2 piperazine), 2.15 (s, 6H, CH3), 1.82 (s,

6416 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Serradji et al.
column with MeOH/CH2Cl2 (1:99, v/v) as eluent to give 6 (2.26
g, 86.8%) as a wax: Rf 0.55 (MeOH/CH2Cl2, 5:95, v/v); IR (ν
cm-1) 3338 (NH), 1730 (CdO amide), 1594 (ArCdC); 1H NMR
(60 MHz) δ 7.20 (br s, 15H, ArH), 5.83 (br s, 1H, NH), 4.00-
3.00 (m, 6H, CH2-NH, CH2-Ph), 2.83-2.18 (m, 7H, CH2 and
CH piperazine).
1,4-Dibenzyl-2-(phenoxycarbonyloxymethyl)piper-
azine (7). This compound was prepared from 5, using the
same process as for 6, as a wax (1.97 g, 88%); Rf 0.31 (MeOH/
CH2Cl2, 5:95, v/v); IR (ν cm-1) 3062 (ArCH), 1758 (CdO), 1599
(ArCdC); 1H NMR δ 7.27-7.00 (m, 15H, ArH), 4.42-4.34 (m,
2H, CH2O), 3.85 (d, 1H, J ) 13 Hz, CH-Ph), 3.44-3.29 (m,
2H, CH2-Ph), 3.32 (d, 1H, J ) 13 Hz, CH-Ph), 2.81-2.79 (m,
1H, CH-CH2O), 2.66-2.50 (m, 2H, piperazine), 2.37-2.22 (m,
4H, piperazine); 13C NMR δ 153.55 (CdO), 138.72, 138.04,
129.37, 128.91, 128.65, 128.18, 127.00, 126.91, 125.93, 120.96
(ArCdC), 67.34, 62.84, 58.55, 55.34, 52.61, 49.48 (CH2 benzyl
and piperazine), 58.08 (CH piperazine).
1,4-Dibenzyl-2-(N,N-diethylaminocarbonylamino-
methyl)piperazine (8a). A solution of 6 (3.2 g, 7.71 mmol)
in Et2NH (10 mL) was refluxed for 20 h. After elimination of
the excess of amine under reduced pressure, the residue was
taken up with CH2Cl2, washed with 1 N NaOH and water,
dried (MgSO4), and filtered, and the solvent was eliminated
in a vacuum. A chromatography on a silica gel column using
MeOH/CH2Cl2 (2:98, v/v) as eluent gave 8a (1.84 g, 61%) as a
wax: Rf 0.29 (MeOH/CH2Cl2, 5:95, v/v); IR (ν cm-1) 3366 (NH),
1636 (CdO ureate), 1589 (ArCdC); 1H NMR δ 7.19 (br s, 10H,
ArH), 5.01 (br s, 1H, NH), 3.94 (d, 1H, J ) 13.6 Hz, CH-Ph),
3.42 (d, 1H, J ) 13 Hz, CH-Ph), 3.33 (d, 1H, J ) 13 Hz, CH-
Ph), 3.24 (d, 1H, J ) 13.6 Hz, CH-Ph), 3.19 (m, 2H, CH2NH),
3.13 (q, 4H, J ) 7 Hz, N(CH2CH3)2), 2.69 (m, 4H, piperazine),
2.19 (m, 3H, piperazine), 1.03 (t, 6H, J ) 7 Hz, N(CH2CH3)2).
purified by crystallization in acetic acid/H2O, as crystals
(870 mg, 53%): Rf 0.33 (NH4OH/MeOH/CH2Cl2, 2:20:80, v/v/
v); mp 134 °C; IR (ν cm-1) 1633 (CdO); 1H NMR (DMSO-d6) δ
6.32 (br s, 1H, NHCdO), 5.14 (br s, 4H, NH2+), 3.10-2.10
(m, 13H, CH2NH, N(CH2CH2CH3)2, piperazine), 1.81 (s, 6H,
CH3COO-), 1.43m (4H, J ) 7.2 Hz, N(CH2CH2CH3)2), 0.80 (t,
6H, J ) 7.2 Hz, CH3); 13C NMR (DMSO-d6) δ 157.24 (CdO),
53.89 (CH piperazine), 47.81, 47.02, 43.42, 42.67, 39.51, 21.31
(CH2 piperazine and ureate), 11.15 (CH3).
2-(N-Butylaminocarbonyloxymethyl)piperazine, Di-
hydrochloride (9c). This compound was prepared as de-
scribed for compounds 3, from 8c (4 g, 10.05 mmol), as crystals
(2.4 g, 82%): Rf 0.14 (NH4OH/MeOH/CH2Cl2, 2:20:80, v/v/v);
mp 188 °C; IR (ν cm-1) 1760 (CdO amide); 1H NMR
(DMSO-d6) δ 10.12 (br s, 4H, NH2+), 7.26 (br s, 1H, NHCdO),
4.22 (m, 2H, CH2OCO), 4.00-2.80 (m, 9H, CH2-NH, pipera-
zine), 1.30 (m, 4H, (CH2)2), 0.84 (t, 3H, J ) 13 Hz, CH3); 13C
NMR (DMSO-d6) δ 155.28 (CdO), 63.80 (CH2O), 50.79 (CH
piperazine), 41.33, 40.07 (CH2 piperazine), 39.32, 31.38, 19.42
(CH2 carbamate), 13.67 (CH3).
2-(N,N-Diethylaminocarbonylaminomethyl)-1,4-bis-
(3,4,5-trimethoxybenzoyl)piperazine (10a). This compound
was prepared as described for compounds 4, from 9a, as
crystals (800 mg, 81%): Rf 0.18 (MeOH/CH2Cl2, 5:95, v/v); mp
148 °C; IR (ν cm-1) 3389 (NH), 1634 (CdO ureate), 1619
(CdO amide), 1584 (ArCdC); 1H NMR δ 6.61 (br s, 4H, ArH),
4.95 (br s, 1H, NH), 4.14 (m, 3H, piperazine), 3.82 and 3.79
(2s, 18H, CH3O), 3.60-2.60 (m, 10H, CH2NH, N(CH2CH3)2,
piperazine), 1.03 (t, 6H, J ) 7 Hz, CH3); 13C NMR δ 171.43,
170.95 (CdO), 157.04 (CdO ureate), 153.39, 153.29, 139.48,
130.28, 130.06, 104.41 (ArCdC), 60.81, 56.31, 56.22 (CH3O),
49.81, 47.10, 43.45, 39.51, 41.11 (CH2), 13.78 (CH3). Anal.
(C30H42N4O9‚0.75H2O) C, H, N.
1,4-Dibenzyl-2-(N,N-dipropylaminocarbonylamino-
methyl)piperazine (8b). This compound was prepared, using
the same process as for 8a, as a wax (1.97 g, 88%): Rf 0.31
(MeOH/CH2Cl2, 5:95, v/v); IR (ν cm-1) 3379 (NH), 1636 (CdO
ureate), 1590 (ArCdC); 1H NMR δ 7.18 (br s, 10H, ArH), 5.05
(t, 1H, J ) 4 Hz, NH), 3.95 (d, 1H, J ) 13.5 Hz, CH-Ph), 3.41
(d, 1H, J ) 13.1 Hz, CH-Ph), 3.32 (d, 1H, J ) 13.1 Hz,
CH-Ph), 3.02 (m, 6H, CH2NH, N(CH2CH2CH3)2), 2.80-2.35
(m, 4H, piperazine), 2.35-1.90 (m, 3H, piperazine), 1.46 (m,
4H, J ) 7.4 Hz, N(CH2CH2CH3)2), 0.80 (t, 6H, J ) 7.4 Hz,
N(CH2CH2CH3)2); 13C NMR δ 157.57 (CdO), 138.41, 137.49,
128.93, 128.35, 128.12, 127.99, 126.88, 126.78 (ArCdC), 62.84,
57.26, 55.69, 52.34, 50.33 (CH2 benyl and piperazine), 58,21
(CH piperazine), 48.87, 40.06, 21.61 (CH2 ureate), 11.18 (CH3).
2-(N,N-Dipropylaminocarbonylaminomethyl)-1,4-bis-
(3,4,5-trimethoxybenzoyl)piperazine (10b). This com-
pound was prepared as described for compounds 4, from 9b,
as crystals (500 mg, 61%): Rf 0.50 (MeOH/CH2Cl2, 5:95, v/v);
mp 78.2 °C; IR (ν cm-1) 3395 (NH), 1636 (CdO ureate), 1617
(CdO amide), 1584 (ArCdC); 1H NMR δ 6.60 and 6.58 (s, 4H,
ArH), 4.88 (br s, 1H, NH), 4.50-3.95 (m, 3H, piperazine),
3.82 and 3.79 (2s, 18H, CH3O), 3.60-3.30 (m, 10H, CH2NH,
N(CH2CH2CH3)2, piperazine), 1.47 (m, 4H, J ) 7.2 Hz,
N(CH2CH2CH3)2), 0.81 (t, 6H, J ) 7.2 Hz, CH3); 13C NMR δ
171.43, 170.95 (CdO amide), 157.43 (CdO ureate), 153.39,
153.29, 139.51, 130.31, 130.07, 104.47 (ArCdC), 60.83, 56.31,
56.23 (CH3O), 49.82, 43.40, 39.75, 49.01, 21.70 (CH2), 11.27
(CH3). Anal. (C32H46N4O9‚H2O) C, H, N.
1,4-Dibenzyl-2-(N-butylaminocarbonyloxymethyl)-
piperazine (8c). This compound was prepared from 7 (5 g,
12 mmol), using the same process as for 8a, as a wax (4 g,
84%): Rf 0.24 (MeOH/CH2Cl2, 3:97, v/v); IR (ν cm-1) 3339 (NH),
2-(N-Butylaminocarbonyloxymethyl)-1,4-bis(3,4,5-tri-
methoxybenzoyl)piperazine (10c). This compound was
prepared as described for compounds 4, from 9c, as crystals
(610 mg, 29%): Rf 0.36 (MeOH/CH2Cl2, 5:95, v/v); mp 101.4
°C; IR (ν cm-1) 3390 (NH), 1717 (CdO carbamate), 1620
(CdO amide), 1586 (ArCdC); 1H NMR δ 6.58 and 6.57 (s, 4H,
ArH), 4.74 (br s, 1H, NH), 4.60-3.90 (m, 5H, CH2O, pipera-
zine), 3.80-3.79 (s, 18H, CH3O), 3.60-2.60 (m, 6H, CH2NH,
piperazine), 1.28 (m, 4H, NHCH2(CH2)2CH3), 0.82 (t, 3H, J )
7 Hz, CH3); 13C NMR δ 170.83 (CdO amide), 155.65 (CdO
carbamate), 153.27, 139.43, 130.16, 130.01, 104.33 (ArCdC),
60.89 (CH2O), 60.74, 56.17 (CH3O), 40.71, 31.76, 19.69 (CH2),
13.51 (CH3). Anal. (C30H41N3O10‚0.5H2O) C, H, N.
1
1712 (CdO amide), 1602 (ArCdC); H NMR δ 7.17 (m, 10H,
ArH), 4.68 (br s, 1H, NH), 4.25 (dd, 1H, J ) 11.3 and 3.8 Hz,
CHOCdO), 4.20 (dd, 1H, J ) 11.3 and 4.5 Hz, CH-Ph), 3.92
(d, 1H, J ) 13.5 Hz, CH-Ph), 3.06 (m, 2H, NH-CH2), 2.80-
1.90 (m, 7H, piperazine), 1.29 (m, 4H, NH-CH2(CH2)2-CH3),
0.84 (t, 3H, J ) 7 Hz, CH3); 13C NMR δ 156.31 (CdO), 138.62,
138.03, 128.88, 128.04, 126.76 (ArCdC), 63.63, 62.81, 58.34,
55.70, 52.66, 49.93 (CH2 benzyl and piperazine), 19.74 (CH2),
13.61 (CH3).
2-(N,N-Diethylaminocarbonylaminomethyl)pip-
erazine, Dihydrochloride (9a). This compound was pre-
pared as described for compounds 3 from 8a (1.48 g, 4.67
mmol) and purified by crystallization in MeOH/Et2O, as
crystals (1.34 g, 35%): Rf 0.1 (NH4OH/MeOH/CH2Cl2, 2:20:80,
v/v/v); mp 103 °C; IR (ν cm-1) 1617 (CdO); 1H NMR
(DMSO-d6) δ 9.95 (br s, 4H, NH2+), 6.71 (br s, 1H, NH), 3.90-
2.80 (m, 13H, CH2NH, N(CH2CH3)2, piperazine), 1.01 (t, 6H,
J ) 6.7 Hz, CH3); 13C NMR (DMSO-d6) δ 156.95 (CdO), 52.22
(CH piperazine), 42.29, 40.33, 39.92 (CH2 piperazine), 39.30
(CH2), 13.87 (CH3).
2-Ethyloxycarbonylpiperazine, Dihydrochloride (12).
This compound was prepared from 11 (21 g, 62.13 mmol) as
described for compound 3a but using 12 N HCl instead of
glacial acetic acid, as a solid (14 g, 97%): Rf 0.39 (NH4OH/
MeOH/CH2Cl2, 2:20:80, v/v/v); mp 199 °C; IR (ν cm-1) 1756
(CdO); H NMR (DMSO-d6) δ 9.74 (br s, 4H, NH2+), 4.56 (m,
1
1H, piperazine), 4.11 (m, 2H, CH2OCdO), 2.80-2.60 (m, 6H,
piperazine), 1.20 (br s, 3H, CH3); 13C NMR (DMSO-d6) δ 156.03
(CdO), 62.73 (CH2O), 51.70 (CH piperazine), 13.86 (CH3).
2-Ethyloxycarbonyl-1,4-bis(3,4,5-trimethoxybenzoyl)-
piperazine (13). This compound was prepared using the same
process as for 4a, from 12, as a solid (13.1 g, 39.6%): Rf
0.4 (MeOH/CH2Cl2, 3:97, v/v); mp 122 °C; IR (ν cm-1) 1735
(CdO ester), 1635 (CdO amide), 1586 (ArCdC); 1H NMR δ
2-(N,N-Dipropylaminocarbonylaminomethyl)pip-
erazine, Diacetate (9b). This compound was prepared as
described for compounds 3 from 8b (1.92 g, 4.55 mmol) and

PAF Antagonists with Anti-HIV-1 Activity
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6417
6.59 (s, 4H, ArH), 5.40-4.30 (m, 3H, piperazine), 4.15 (m, 2H,
CH2OCdO), 3.81 and 3.79 (2s, 18H, OCH3), 3.60-2.60 (m, 4H,
piperazine), 1.19 (t, 3H, J ) 7 Hz, OCH2CH3); 13C NMR δ
171.13, 170.54 (CdO amide), 169.08 (CdO ester), 153.32,
153.14, 139.51, 129.81, 104.52, 104.13 (ArCdC), 61.86 (CH2O),
60.72, 56.12 (CH3O), 52.32 (CH piperazine), 44.95 (CH2 pip-
erazine), 13.91 (CH3).
2-Hydroxymethyl-1,4-bis(3,4,5-trimethoxybenzoyl)pip-
erazine (14). NaBH4 (7 g, 183.1 mmol) was added portionwise
to a cooled (ice bath) solution of 13 (10 g, 18.31 mmol) in MeOH
(100 mL). The solution was stirred for 1 h at 0 °C and overnight
at room temperature. The solvent was then evaporated and
the residue taken up in CH2Cl2. The solution was washed
several times with water, dried over MgSO4, and filtered, and
the solvent was eliminated under reduced pressure. The
compound was crystallized in MeOH/Et2O, and 6.50 g (70%)
of white crystals was obtained: Rf 0.45 (MeOH/CH2Cl2,
10:90, v/v); mp 139 °C; IR (ν cm-1) 3370 (OH), 1636 (CdO),
1581 (ArCdC); 1H NMR δ 6.59 (s, 4H, ArH), 5.25-3.90 (m,
4H, CH2O, OH, piperazine), 3.80 and 3.79 (s, 18H, CH3O),
3.70-2.70 (m, 6H, piperazine); 13C NMR δ 171.51 (CdO),
153.23, 139.60, 139.24, 130.23, 129.47, 104.52, 104.14
(ArCdC), 60.73, 56.11 (CH3O), 59.16 (CH2OH).
2-(N,N-Diethylthionocarbamoyloxymethyl)-1,4-bis(3,4,5-
trimethoxybenzoyl)piperazine (15a). To a suspension of
NaH (60% in mineral oil, 100 mg, 2.4 mmol) in DMF (5 mL)
was added dropwise a solution of the alcohol 14 (1 g, 2 mmol)
in DMF (10 mL). When the alcoholate was formed, a solution
of N,N-diethylthiocarbamoyle chloride (370 mg, 2.4 mmol) in
DMF (10 mL) was added dropwise to the former suspension.
The mixture was heated at the end of the addition to 70-80
°C and stirred for 23 h. Then saturated aqueous NaCl (50 mL)
was added, the reaction mixture was extracted with EtOAc (3
× 50 mL) and dried over MgSO4, and the solvent was removed
in a vacuum. The residue was chromatographed using a silica
gel column with MeOH/CH2Cl2 (1:99, v/v) as eluent and
crystallized in MeOH/EtOH to yield the title compound as
yellow crystals (800 mg, 65%): Rf 0.43 (MeOH/CH2Cl2, 5:95,
v/v); mp 175.3 °C; IR (ν cm-1) 3045 (ArH), 1614 (CdO), 1582
(ArCdC); 1H NMR δ 6.60 (s, 2H, ArH), 6.59 (s, 2H, ArH), 5.40-
4.60 and 4.60-4.00 (m, 5H, CH2OCdS, piperazine), 3.82, 3.80
and 3.79 (3s, 18H, CH3O), 3.75-3.10 (m, 4H, SdCN(CH2CH3)2,
piperazine), 3.41 (q, 2H, J ) 7 Hz, SdCN(CH2CH3)2), 1.14 and
0.98 (t, 6H, J ) 7 Hz, CH3); 13C NMR δ 186.30 (CdS), 170.84,
170.71 (CdO), 153.41, 153.28, 138.48, 139.30, 130.12, 104.43,
104.05 (ArCdC), 66.53 (CH2O), 60.78, 56.29 (CH3O), 47.86,
43.31 (CH2), 13.20, 11.80 (CH3). Anal. (C30H41N3O9S‚0.5H2O)
C, H, N.
CH2CH3), 1.00 (t, 3H, J ) 7 Hz, CH2CH3); 13C NMR δ.173.73
(CdO ester), 170.69 (CdO), 153.25, 139.41, 130.04, 129.86,
104.17 (ArCdC), 59.94 (CH2O), 60.68, 56.07 (CH3O), 27.10
(CH2), 8.69 (CH3). Anal. (C28H36N2O10) C, H, N.
3-(N,N-Diethylaminocarbonyloxymethyl)-1-(triphenyl-
methyl)piperazine (17). To a solution of 16 (6.26 g, 21.73
mmol) and Et3N (12 mL, 86.92 mmol) in CH2Cl2 (100 mL) was
added dropwise a solution of triphenylmethyl chloride (6 g,
21.73 mmol) in CH2Cl2 (100 mL). The solution was stirred for
6 h and was then washed with saturated NaHCO3 solution
and water until neutral pH, dried (MgSO4), and filtered, and
the solvent was eliminated in a vacuum. The residue (9.9 g,
99.7%) was used for next steps without any further purifica-
tion: Rf 0.093 (MeOH/CH2Cl2, 3:97, v/v); IR (ν cm-1) 3320 (NH),
3045 (ArCH), 1695 (CdO carbamate), 1595 (ArCdC); 1H NMR
δ 7.40 (m, 6H, ArH), 7.10 (m, 9H, ArH), 3.86 (m, 2H, CH2O),
3.50-2.45 (m, 8H, N(CH2CH3)2), 2.00-1.10 (m, 4H, pipera-
zine), 0.97 (m, 6H, N(CH2CH3)2); 13C NMR δ 155.46 (CdO),
129.26, 127.40, 125.91 (ArCdC), 66.85 (CH2O), 54.68 (CH
piperazine), 51.50, 48.78, 45.75 (CH2 piperazine), 41.67, 41.21
(NCH2CH3), 13.93, 13.45 (NCH2CH3)
2-(N,N-Diethylaminocarbonyloxymethyl)-1-(3,4,5-tri-
methoxybenzoyl)-4-(triphenylmethyl)piperazine (18). To
a stirred solution of 17 (7.5 g, 16.45 mmol) and Et3N (7 mL,
49.35 mmol) in CH2Cl2 (60 mL) was added dropwise a solution
of 3,4,5-trimethoxybenzoyl chloride (4.44 g, 19.25 mmol) in
CH2Cl2 (50 mL). The mixture was stirred overnight at room
temperature and then washed with a saturated NaHCO3
solution and water. The organic layer was dried over MgSO4,
filtered, and concentrated in a vacuum. Purification on a silica
gel column using CH2Cl2 as eluent yielded 18 (9.52 g, 89%) as
a wax: Rf 0.33 (MeOH/CH2Cl2, 3:97, v/v); IR (ν cm-1) 1701
(CdO carbamate), 1616 (CdO amide), 1507 (ArCdC); 1H NMR
δ 7.38 and 7.19 (m, 15H, ArH), 6.49 (s, 2H, ArCH), 4.99 and
4.41 (m, 2H, piperazine), 3.83 (m, 2H, CH2O), 3.74 and 3.73
(s, 9H, CH3O), 3.59 (q, 2H, J ) 7 Hz, NCH2CH3), 3.60-2.60
(m, 6H, N(CH2CH3)2, piperazine), 1.79 (m, 1H, piperazine), 1.09
(m, 6H, CH3); 13C NMR 169.97 (CdO amide), 155.78 (CdO
carbamate), 152.96, 138.96, 130.86, 129.13, 127.53, 126.20,
104.31 (ArCdC), 62.83 (CH2O), 60.66, 56.02 (CH3O), 49.25 (CH
piperazine), 48.97, 48.00, 44.93 (CH2 piperazine), 41.69, 41.12
(NCH2CH3), 13.90, 13.43 (NCH2CH3).
2-(N,N-Diethylaminocarbonyloxymethyl)-1-(3,4,5-tri-
methoxybenzoyl)piperazine (19). To a solution of 18 (6.5
g, 9.98 mmol) in MeOH (100 mL) was added dropwise 12 N
HCl (6 mL). The solution was stirred for 10 min at room
temperature and the solvent was removed in a vacuum. The
residue was then taken up with CH2Cl2, washed with a
saturated NaHCO3 solution and water, dried (MgSO4), filtered,
and concentrated. A rapid purification on a silica gel column
using MeOH/CH2Cl2 as eluent (3:97, v/v) yielded 19 (3.8 g,
2-(N,N-Dimethylthionocarbamoyloxymethyl)-1,4-bis-
(3,4,5-trimethoxybenzoyl)piperazine (15b). Compound 15b
(1.2 g, 51%) was prepared as a solid from the alcohol 14 and
N,N-dimethylthiocarbamoyle chloride as described for com-
pound 15a: Rf 0.24 (MeOH/CH2Cl2, 3:97, v/v); mp 171.3 °C;
93%) as a wax: Rf 0.11 (MeOH/CH2Cl2, 3:97, v/v); IR (ν cm-1
)
3330 (NH), 1693 (CdO carbamate), 1632 (CdO amide), 1584
(ArCdC); 1H NMR δ 6.55 (s, 2H, ArH), 4.90-4.40 (m, 2H,
piperazine), 4.29 (m, 2H, CH2O), 3.79 and 3.77 (s, 9H, CH3O),
3.40-2.50 (m, 9H, N(CH2CH3)2, piperazine), 1.37 (br s, 1H,
NH), 1.02 (t, 6H, J ) 7 Hz, CH3); 13C NMR δ 170.44 (CdO
amide), 155.36 (CdO carbamate), 153.07, 138.95, 130.94,
104.08 (ArCdC), 61.44 (CH2O), 60.62, 56.10 (CH3O), 45.86 (CH
piperazine), 41.67, 41.07 (NCH2CH3), 13.88, 13.25 (NCH2CH3).
1
IR (ν cm-1) 1623 (CdO), 1582 (ArCdC); H NMR δ 6.59 and
6.57 (s, 4H, ArH), 5.40-3.90 (m, 5H, CH2OCdS, piperazine),
3.82, 3.80 and 3.79 (3s, 18H, CH3O), 3.70-3.35 (m, 2H,
piperazine), 3.25 (s, 3H, CH3), 3.20-2.30 (m, 5H, piperazine);
13C NMR δ 186.88 (CdS), 170.65 (CdO), 153.23, 139.32,
139.10, 130.00, 104.15, 103.89 (ArCdC), 66.38 (CH2O), 60.64,
56.18, 56.14 (CH3O), 42.67, 37.53 (CH3). Anal. (C28H37N3O9S)
C, H, N.
2-(N,N-Diethylaminocarbonyloxymethyl)-4-(4-hydroxy-
3,5-dimethoxybenzoyl)-1-(3,4,5-trimethoxybenzoyl)pip-
erazine (20). A mixture of 19 (3 g, 7.33 mmol), syringic acid
(1.5 g, 8.06 mmol), DCC (1.55 g, 8.06 mmol), and HOBT (1.2
g, 8.06 mmol) in CH2Cl2 (25 mL) was refluxed overnight. The
solution was then filtered; washed with 1 N HCl, water, a
saturated NaHCO3 solution, and water; and dried over MgSO4.
After the evaporation of the solvent, purification on a silica
gel column using MeOH/CH2Cl2 as eluent (1:99, v/v) and
crystallization in MeOH/Et2O yielded 20 (3.67 g, 84.5%) as
crystals: Rf 0.21 (MeOH/CH2Cl2, 5:95, v/v); mp 129.3 °C; IR (ν
cm-1) 3373 (OH), 1686 (CdO carbamate), 1617 (CdO amide),
1586 (ArCdC); 1H NMR δ 6.61 (s, 2H, ArH), 6.57 (s, 2H, ArH),
5.69 (s, 1H, OH), 5.3-3.9 (m, 5H, CH2O and piperazine), 3.80
2-(Ethylcarbonyloxymethyl)-1,4-bis(3,4,5-trimethoxy-
benzoyl)piperazine (15c). To a solution of the alcohol 14
(500 mg, 1 mmol) and Et3N (140 µL, 2 mmol) in CH2Cl2 (10
mL) was added dropwise propanoyl chloride (208 µL, 4 mmol)
in CH2Cl2 (5 mL). After stirring overnight at room tempera-
ture, the solution was washed with water, dried over MgSO4,
and concentrated in a vacuum. Purification on a silica gel
column using CH2Cl2 as eluent and crystallization in MeOH/
ether yielded 15c (460 mg, 82%) as crystals: Rf 0.39 (MeOH/
CH2Cl2, 5:95, v/v); mp 139.6 °C; IR (ν cm-1) 2954 (ArCH), 1746
(CdO ester), 1630 (CdO amide), 1586 (ArCdC); 1H NMR δ
6.55 (s, 4H, ArH), 5.20-3.95 (m, 5H, CH2O, piperazine), 3.80
(br s, 18H, CH3O), 3.50-2.60 (m, 4H, piperazine), 2.16 (m, 2H,

6418 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Serradji et al.
(s, 15H, CH3O), 3.6-2.4 (m, 8H, NCH2CH3 and piperazine),
1.4-0.65 (m, 6H, CH3); 13C NMR δ.171.06, 170.62 (CdO
amide), 155.05 (CdO amide), 153.26, 146.91, 139.46, 136.58,
130.21, 125.35, 104.42, 104.25 (ArCdC), 60.89 (CH2O), 60.76,
56.38, 56.15 (CH3O), 41.72, 41.01 (NCH2CH3), 13.80, 13.29
(NCH2CH3). Anal. (C29H39N3O10‚1.5H2O) C, H, N.
in an infusion pump (Harvard pump PHD 2000, Harvard
Apparatus, Holliston, MA) and connected to the catheter.
Before perfusion, the thorax of the animal was opened, the
heart cut, and perfusion immediately started with a flow rate
of 2.5 mL/min. The perfusion fluid consisted of bicarbonate-
buffered physiological saline (mM): 128 NaCl, 24 NaHCO3, 4.2
KCl, 2.4 NaH2PO4, 1.5 CaCl2, 0.9 MgCl2, and 9 D-glucose. The
solution was gassed with 95% O2 and 5% CO2 for pH control
(7.4) and warmed to 37 °C in a water bath. Tracers were added
to the perfusate at a concentration of 0.3-0.4 µCi/mL. Perfu-
sion was terminated by decapitation after 60 s. The brain was
removed from the skull and dissected on ice. The right cerebral
hemisphere was placed in tared vials and weighed. Aliquots
of the perfusion fluid were also collected and weighed to
determine tracer concentrations in the perfusate. Samples
were digested in 1 mL of Solvable (Packard, Rungis, France)
at 50 °C and mixed with 9 mL of Ultima gold XR (Packard).
Dual label counting was performed simultaneously in a
Packard Tri-Carb model 1900 TR (Packard).
3H-Labeling. To a solution of 20 (1.31 × 10-4 M, 1.1 equiv)
and K2CO3 in toluene (1 mL) was added dropwise a solution
of [3H]CH2I (10 mCi/mL, 1 mL, Amersham) in toluene. After
the mixture was stirred at room temperature for 24 h and the
addition of a few milliliters of CH3CN, the resulting solution
was stirred again for 30 h. Two compounds appeared after UV-
detection on a TLC plate: the precursor 20 (Rf 0.21 in MeOH/
CH2Cl2, 5:95, v/v) and the final compound [3H]1a (compound
21, Rf 0.48 in MeOH/CH2Cl2, 5:95, v/v). The solvent and traces
of [3H]CH2I were then removed with a stream of N2, and the
residue taken up with CH2Cl2 was chromatographed using a
TLC plate (MeOH/CH2Cl2, 3:97, v/v as eluent). Autoradiogra-
phy (BIOMAX MR, Kodak) was then performed (3-h exposure
in a cassette), the radioactive band corresponding to 1a (Rf (
0.05) was cut, and the silica was recovered and extracted
several times with MeOH/CH2Cl2 (10:90, v/v) to yield 10 µCi
of [3H]1a.
Calculation of Blood-Brain Transport Parameters.
The brain uptake was expressed as a blood-brain transfer
coefficient Kin (µL/s/g) and was calculated from
Kin ) Vbrain/T
(1)
Biological Methods. Platelet Aggregation. The inhibi-
tion of platelet aggregation was conducted according to the
published procedures.12 Briefly, it was determined using
platelet-rich plasma (PRP) of New Zealand rabbits by the
method of Cazenave et al.22 Blood samples were collected from
the auricular artery into a citrate buffer (3.8%, pH 7.4), and
PRP was obtained by centrifugation for 15 min at 1200 rpm.
The antagonists were solubilized in EtOH at concentrations
from 10-2 to 10-7 M and added to the incubated and stirred
PRP for 2 min before PAF (2.5 nM) challenge. Platelet
aggregation induced by PAF in the presence of the antagonists
was monitored by continuous recording of light transmission
in a dual-channel recorder (Cronolog Coultronics Apparatus)
and was compared to a control aggregation induced by PAF
alone. The drug concentration required to produce 50% inhibi-
tion (IC50) was calculated from dose-response curves (five or
six determinations).
where T is the perfusion time (s).
The apparent volume of the distribution (Vbrain) was calcu-
lated from the amount of radioactivity in the right brain
hemisphere using the following equation
Vbrain ) Xbrain/Cperf
(2)
where Xbrain (dpm/g) is the calculated amount of [3H]1a in the
right cerebral hemisphere and Cperf (dpm/µL) is the [3H]tracer
concentration in the perfusion fluid.
Brain tissue radioactivity was corrected for vascular con-
tamination (Vvasc) with the following equation
Xbrain ) Xtot - VvascCperf
(3)
where Xtot (dpm/g) is the total quantity of tracer measured in
the tissue sample (vascular + extravascular).
Brain vascular volume (Vvasc; µL/g) was estimated from the
tissue distribution of [14C]sucrose, which is known to diffuse
very slowly across the BBB, using the following equation
Antiviral Assay. Antiviral assays and data analysis were
conducted according to published procedures.12,13 HIV-1 rep-
lication was assessed in cell culture supernatants by quan-
tifying reverse transcriptase (RT), using the RetroSys kit
(Innovagen).
Mouse Brain Perfusion Technique. Materials. [14C]-
sucrose (565 mCi/mmol) was purchased from Amersham
Pharmacia Biotech (Orsay, France). Heparin sodium was
obtained from Sanofi & Synthelabo (Gentilly, France). All other
chemicals were commercial products of analytical grade.
Animals. Adult male OF-1 mice (30-40 g, 6-8 weeks old)
were obtained from Iffa Credo (L’arbesle, France). Animals
were housed in a room with a controlled environment (22 ( 3
°C; 55 ( 10% relative humidity) and maintained under a 12 h
dark-light cycle (light from 6:00 a.m. to 6:00 p.m.). Animals
had access to food and tap water ad libitum. All animals
procedures complied with the Principles of Laboratory Animal
Care (NIH publication # 85-23, revised 1985).
Surgical Procedures and Transport Studies. Mice were
anaesthetized by intraperitoneal injection of xylazine (Bayer,
Puteaux, France; 8 mg/kg) and ketamine (Parke Davis, Cour-
bevoie, France; 140 mg/kg). Blood-brain transport of the
compounds was measured in mice using the in situ brain
perfusion technique recently described.15 Briefly, the right
common carotid was catheterized with polyethylene tubing
(0.30 mm i.d. × 0.70 mm o.d., Biotrol Diagnostic, Chennevi-
e`res-les-Louvre, France) that was filled with heparin (25
U/mL) and mounted on a 26 gauge needle. Before insertion of
the catheter, the common carotid artery was ligated caudally.
The external carotid was ligated rostral to the occipital artery
at the level of the bifurcation of the common carotid artery.
During surgery, body temperature was maintained from 37
to 38 °C using a rectal thermistor connected to a temperature
monitor. The syringe containing the perfusion fluid was placed
Vvasc ) X*/Cperf
*
(4)
where X* (dpm/g) is the amount of sucrose measured in the
right brain hemisphere and Cperf* (dpm/µL) is the concentration
of the labeled sucrose in the perfusion fluid.
Data are presented as mean ( SD for five animals.
Acknowledgment. The authors thank BIOTEC
CENTRE (Orle´ans, France) for the pharmacokinetic
study of 1a and the Centre International de Toxicologie
(CIT, Evreux, France) for the acute oral toxicity experi-
ment. This work was supported by the Scientific Concil
of the Universite´ Paris 7sDenis Diderot (Paris, France),
the Fondation pour la recherche Me´dicale-SIDACTION
(Paris, France), and the Comite´ Mixte d′Evaluation et
de Prospection de Coope´ration Interuniversitaire Franco-
Alge´rienne (CMEP n°00 MDU 477).
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